Recombinant mva strains as potential vaccines against p. falciparum malaria

ABSTRACT

This invention relates to recombinant viruses based on MVA, which comprise at least one nucleic acid coding for a  Plasmodium falciparum  MSP-1 protein, a fragment or mutein of it. Furthermore, methods for the production of the recombinant viruses, virus-containing vaccines and the use of the recombinant viruses for the prophylaxis and/or therapy of malaria are provided.

CROSS-REFERENCE

This application is a national stage filing under 35 U.S.C.§371 ofInternational Patent Application Serial No. PCT/EP2003/010723, which wasfiled on Sep. 26, 2003 and which was published as WO 2004/038024 on May6, 2004 which International Patent Application claims benefit ofpriority of German Patent Application no. 10249390.1, filed Oct. 23,2002, which application is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This application is in the field of recombinant vaccinia virus, and inthe field of vaccines to Plasmodium falciparum malaria.

BACKGROUND OF THE INVENTION

The invention relates to the production of recombinant vaccinia virusesof the strain MVA (Modified Vaccinia Virus Ankara) for the recombinantproduction of the complete malaria antigen gp190/MSP-1 of the malariapathogen Plasmodium falciparum as well as individual naturally occurringdomains and parts thereof. Furthermore, the invention relates to the useof recombinant MVA which contain the synthetic DNA sequence of themalaria antigen and parts thereof integrated into the virus genome asvaccines for immunisation against malaria.

Malaria is one of the most dangerous infectious diseases in the world.According to estimates from the World Health Organisation (WHO) 400 to900 million incidences of the disease are registered annually. Accordingto information from the Multilateral Initiative against Malaria (MIM)between 700,000 and 2.7 million people die each year from the infection(MIM, 2001). In this respect 40% of the world's population in 99countries are put at risk from malaria. The disease is caused bysingle-cell protozoa of the genus Plasmodium from the phylumApicomplexa. There are four species which infect humans: Plasmodiummalariae, responsible for Malaria quartana, Plasmodium vivax andPlasmodium ovale, both of which cause Malaria tertiana, and finallyPlasmodium falciparum, the pathogen of Malaria tropica and responsiblefor almost all fatal infections.

It is again currently spreading to an increasing extent. This isprimarily attributable to intensive resistance formation of the malariapathogen which is promoted in that the medicaments used for the therapyare also recommended and used for prophylaxis. Apart from the search fornew chemotherapeutics, research is concentrating on the development ofvaccines, because in the course of malaria infections in humans,immunity mechanisms are applied, a fact which is expressed in anincreased resistance to the plasmodia, as demonstrated in thedevelopment of various types of immunity in humans in regions wheremalaria epidemics prevail.

MSP-1 as Potential Vaccine

MSP-1, the main surface protein of merozoites, the invasive form of theblood phases of the malaria pathogen, is a 190-220 kDa protein. Thisprotein is processed later during the development of the merozoites intosmaller protein fragments, which can be present and isolated up to theinvasion of erythrocytes due to the parasites anchored as a complex viaa glycosylphosphatidylinositol anchor on the merozoite surface.

The sequences of the MSP-1 proteins of various P. falciparum strainsfall into two groups, which have been named after two representativeisolates K_(i) and MAD20. Overall the protein consists of a number ofhighly preserved regions, of dimorphous regions, each of which can beassigned to one of the two relatively small oligomorphous blocks in theN-terminal part of the protein (FIG. 1; Tanabe et al., 1987).

The immunisation of mice with the protein purified from P. yoeliiparasites protected the animals from the otherwise fatal infection(Holder and Freeman, 1981). Also the transfer of monoclonal antibodiesagainst MSP-1 from P. yoelii provided protection in the mouse model(Majarian et al., 1984).

Apart from studies on mice, Saimiri and Aotus monkeys have also beenimmunised with native, immune-affinity purified MSP-1. In these teststhe protein obtained from P. falciparum partially (Perrin et al., 1984)resp. completely (Siddiqui et al., 1987) protected against the ensuinginfection with the parasite.

Purification of native material from Plasmodia is however expensive andcannot be used for production on a large scale. Therefore research intovaccines is concentrated on the development of recombinant vaccines.

For example, mice have been successfully immunised with MSP-1-19purified from E. coli or Saccharomyces cerevisiae (Daly and Long, 1993;Ling et al., 1994; Tian et al., 1996; Hirunpetcharat et al., 1997),similarly as Mycobacterium bovis carrying MSP-1-19 (Matsumoto et al.,1999). Alternatively to immunisation with native or recombinantproteins, DNA coding for MSP-1-19 has also been used as a vaccine andprotected immunised mice against infection with P. chabaudi (Wunderlichet al., 2000).

The immunisation of monkeys with recombinant MSP-1-19 and MSP-1-42 fromP. falciparum provided partial protection (Kumar et al., 1995; Egan etal., 2000; Chang et al., 1996; Stowers et al., 2001). The interpretationof immunisation experiments on monkeys is however only conditionallypossible, because a statistical evaluation of the results does not arisedue to the low number of animals in the experiment.

In Phase I and II studies with MSP-1 fragments as vaccine theirimmunogeneity was also shown in humans. In this respect p19 from P.falciparum fused on a T-helper cell epitope of tetanus toxin (Keitel etal., 1999) and the MSP-1 blocks 3 and 4 (Saul et al., 1999; Genton etal., 2000) are involved.

Some epidemiological studies in endemic regions show with adults acorrelation between antibody titers against MSP-1 and the immunityagainst malaria (Tolle et al., 1993; Riley et al., 1992; Riley et al.,1993).

These investigations together with the immunisation studies on animalsprove that MSP-1 is a promising candidate for the development of amalaria vaccine.

Generally, these studies can be differentiated into two approaches;either purified material from parasites or material obtained inheterologic systems was used.

Both for functional investigations and also for use as a vaccine,proteins must be produced reproducibly in a good yield and high quality.MSP-1 can be purified from parasites, but this is only possible on asmall scale and with great expense and therefore cannot be carried outfor obtaining MSP-1 according to the stated criteria in this way.

Vaccinia viruses belong to the genus Orthopoxvirus in the branchChordopoxyirinae. With the pox viruses complex viruses are involvedwhich, with a double-strand DNA genome of about 200 kb and a size of250×350 nm, are some of the largest known viruses. They consist of acuboid shaped virion which is enclosed in a membrane envelope. In thehost cell, replication and generation of the pox viruses takes placeexclusively in the cytoplasm (for an overview: Moss et al., 1996). Here,Vaccinia viruses possess a very wide host cell spectrum and they infectalmost all cells both from humans and also animals. In 1953 Anton Mayrisolated and purified the dermovaccinia strain Chorioallantois VacciniaAnkara (CVA). This virus was propagated further with continuous passageon chicken embryo fibroblasts and an attenuated virus was obtained,which did not show any further virulence in animals and humans (Sticklet al., 1974). Irrespective of this, the virus could be further used forimmunoprophylaxis against diseases caused by orthopox viruses in humansand animals (Stickl et al., 1974). This virus was named ModifiedVaccinia Virus Ankara (MVA) after its location of origin.

Considered on a molecular genetic level, during over 570 passages onchicken embryo fibroblasts the virus lost 31 kb of DNA sequence of itsgenome, principally in the form of six larger deletions, including atleast two genes which determine the host spectrum and therefore thecapability of the virus to replicate (Meyer et al., 1991). During MVAinfection in most of the cells originating in mammals, including humancells, the formation of infectious virus particles is blocked only verylate in the infection cycle at the phase of virion formation, i.e. viralgenes under the control of promoters, both for the early and alsointermediate and late transcription, can be expressed even innon-permissive cells. This differentiates MVA from other attenuated andreplication-deficient pox viruses, such as for example, Vaccinia virusNYVAC or canary pox virus ALVAC, the infection of which is interruptedin most cells originating from mammals already before the viral DNAreplication (Tartaglia et al., 1992; Sutter and Moss, 1992).

In the development of malaria vaccines various recombinant Vacciniaviruses have been used and in this respect replication-competent virusesof the type Western Reserve and Copenhagen and attenuated viruses of thetypes NYVAC, ALVAC or COPAC have been used (Kaslow, et al., 1991;Etlinger and Altenburger, 1991; Aidoo et al., 1997; Allsopp et al.,1996).

In connection with the attenuated Vaccinia virus MVA, only recombinantviruses have been described which carry CSP from the rodent parasitePlasmodium berghei as malaria antigen (Schneider et al., 1998; Plebanskiet al., 1998; Degano et al., 1999; Gilbert et al., 1999).

Furthermore, recombinant Vaccinia viruses are described, which containan MSP-1 coding sequence. The authors of one publication state that theyhave integrated the native MSP-1 coding sequence into the genome of thevirus type Western Reserve, but do not support this statementexperimentally (no restriction analyses, PCR, Northern Blot and WesternBlot analyses, etc.). An immunisation of Saimiri monkeys with theserecombinant viruses did not lead to the formation of MSP-1 specificantibodies and moreover also remained without measurable influence onthe humoral immune response against MSP-1 after a P. falciparuminfection (Pye et al., 1991).

In a further publication the vector NYVAC-Pf7 is described, which, amongother things, expresses msp-1 of P. falciparum. The sera of two of thesix immunised Rhesus monkeys show in the Western Blot analysis none ofthe signals against native MSP-1 detectable in the publication. Thesignals from three further animals detect solely parts of the proteincomplex and only the serum of one of the immunised animals detects abroader band spectrum. Overall however, these signals also appear to beweak. There is no reference to quantitative analyses on MSP-1 specificantibodies by ELISA (Tine et al., 1996).

In human experiments of the Phase I/IIa with NYVAC-Pf7 neither acellular immune response against MSP-1 nor a humoral immune responseverifiable by ELISA was proven (Ockenhouse et al., 1998).

In contrast to this, Tine et al. (1996) verify intact MSP-1 in theWestern Blot analysis, whereby it appears to be a contradiction thatMSP-1 is transported out of the cell by P. falciparum signal sequences(cf. publications from Moran and Caras, 1994, Yang et al., 1997).

SUMMARY OF THE INVENTION

This invention relates to recombinant viruses based on MVA, whichcomprise at least one nucleic acid coding for a Plasmodium falciparumMSP-1 protein, a fragment or mutein of it. Furthermore, methods for theproduction of the recombinant viruses, virus-containing vaccines and theuse of the recombinant viruses for the prophylaxis and/or therapy ofmalaria are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the primary structure of the MSP-1 derived from the FCB-1and MDA20 strains of P. falciparum.

FIG. 2 depicts verification of MSP-1D-42 and MSP-1D-38/42 in HeLa cellsinfected with recombinant MVA using immunoblot.

FIGS. 3A and 3B depict development of a humoral response after threeimmunizations with rMVA-msp1d/42S or rMVA-msp1d/42A and one immunizationwith MSP-1 D-HX42 from E. coli.

FIG. 4 depicts development of a humoral immune response afterimmunizations with rMVA-msp1d/S or rMVA-msp1d/83+30/38+42A incombination with immunization with MSP-1D from E. coli.

DETAILED DESCRIPTION OF THE INVENTION

The object of this invention is therefore to provide a recombinantVaccinia virus that is capable of

containing stably integrated DNA sequences, which code for MSP-1 of P.falciparum or partial sections of it,

expressing these sequences efficiently and reproducibly and therefore

produce MSP-1 protein in secreted or surface-anchored form to

immunise a host and thereby

cause a cellular and humoral immune response.

The object of the invention is solved by the provision of a recombinantMVA virus, which is capable of infection, replication and expression ofMSP-1 in a host. Furthermore, methods for the production and use of therecombinant virus are given.

According to the invention, the expression “virus based on MVA”signifies a virus derived from MVA, which exhibits one or more mutationsin non-essential regions of the virus genome. The essential regions ofthe MVA virus are in this regard all genome sections of the MVA virus,which are necessary for receiving the viral gene expression and thecapability of the MVA virus for propagation. This includes, for example,the gene sequences coding for viral RNA polymerase subunits or the viralDNA polymerase. Preferably the virus based on MVA is the MVA virus.

The Vaccinia system NYVAC-Pf7, known from the state of the art, is basedon the basic virus NYVAC, which originally comes from the CopenhagenVaccinia virus strain and was attenuated by the targeted deletion of 18open reading frames. However, with NYVAC in mammal cells the DNAreplication is blocked (Tartaglia et al., 1992), whereas with MVA thevirus assembly is suppressed. This has the advantage that in contrast toNYVAC in MVA, late gene suppression occurs, which can be used for theexpression of recombinant genes. MVA can therefore both preferentiallyinduce a cytotoxic T-cell response during the early transcription phaseas well as stimulate the humoral branch of the immune response due tothe high protein expression during the late phase.

MVA, which has already been extensively employed during the poxprotection vaccination campaign, is regarded as a very safe virus forvaccination on humans (Stickl et al., 1974).

According to the invention, a recombinant virus based on MVA isprovided, comprising at least one nucleic acid encoding a P. falciparumMSP-1 protein, a fragment or mutein thereof.

The MSP-1 amino acid sequence can be obtained from publicly accessibledata bases. 3D7 (MAD20 isolate): CAA84556; FCB-1 (K1 isolate): CAB36903,both from the NIH data base on the internet at ncbi.nlm.nih.gov.

Particularly preferred is the nucleic acid encoding MSP-1 protein, anucleic acid reduced in its AT content, as described in DE 19640817 A1,the disclosure content of which is included hereby. In particular anucleic acid is preferred which is derived from the P. falciparum MSP-1sequence and in which a large part of the plasmodium codons has beenreplaced such that the codon frequency of the synthetic gene correspondsto the human one without the amino acid sequence being changed.

According to a preferred embodiment, the MSP-1 protein is the MSP-1 ofthe isolate 3D7, the MSP-1 protein which is designated in the followingas “MSP-1D”. Alternatively, this can be the MSP-1 protein of the FCB1strain, which is designated as “MSP-1F” in the following. The MSP-1protein preferably comprises also fragments of these two forms of MSP-1.Especially preferred in this respect are, alone or in combination, thefragments of MSP-LF p83, p30, p38, p33 and p19. Especially preferred,alone or in combination, whereby here similarly combinations with MSP-1Ffragments are included, are fragments of MSP-1D, in particular p83, p30,p38, p33 and p19. In particular the combinations of p83 and p30 as wellas p38 and p42 are preferred. The position of the fragments is in thisrespect shown in FIG. 1. Furthermore, the fragment p42 of both MSP-1forms is also included.

The MSP-1 protein can also be a mutein of the P. falciparum MSP-1sequence, which is differentiated from the MSP-1 sequence of the wildtype by addition, deletion, insertion, inversion or substitution of oneor more amino acids.

In a preferred embodiment the virus according to the invention comprisesa promoter suitable for expression, whereby the sequence encoding msp-1is under the control of the promoter. The promoter can in this respectbe an MVA promoter, whereby the promoter can be an early, intermediateor late gene promoter or a combination of them. However, non-MVApromoters are also included which are capable of expression in theexpression system used. In this respect, both constitutive as well asinducible promoters can be used. For large-scale protein production inthis respect, a strong Vaccinia virus promoter, such as the syntheticlate or early/late promoter or the HybridVaccinia/T7 polymerase systemcan be used; for the induction of MHC Class I restricted cytotoxicT-cell response in vivo a naturally occurring early or tandem early/latepromoter can be used; furthermore the E. coli lac repressor/operatorsystem or the HybridVaccinia/T7 system can be used for the initiation ofthe gene expression; (Methods in Molecular Biology, Volume 62, publishedby Rocky S. Tuan, Humana Press, Broder and Earl, page 176, with furtherverification).

According to a further preferred embodiment, the virus also comprises aselection marker. The selection marker is in this respect suitable forthe selection and/or for screening in a known manner. Suitable selectionmarkers comprise in this respect for example the E. coli lacZ system,the selection system using the E. coli xanthine-guanine phosphoribosyltransferase (XGPRT) gene. In addition selection methods can be usedwhich modify the host cell specificity of the viruses (Staib et al.,2000). Other selection markers known in the state of the art can beused.

According to a further preferred embodiment, the nucleic acid is fusedat the 5′ end with a nucleotide sequence encoding a signal peptidesequence. As known from the state of the art, the signal and anchorsequences from P. falciparum are not detected with expression inmammalian cells or are not correctly processed (Moran and Caras, 1994;Burghaus et al., 1999; Yang et al., 1997).

The problem of the selective control of the intracellular gating issolved by the use of the signal sequences of the human “DecayAccelerating Factor” (DAF) (FIG. 2). Suitable signal peptide sequencesare specific for higher eukaryotes. Examples of such signal sequencesapart from those of the decay accelerating factor are immunoglobulins orsignal peptides of various growth factors and cytokines (von Heijne,1985). According to a preferred embodiment the signal peptide sequencecontrols the secretion of the gene product, for example cytokines,antibodies, etc.

Furthermore, signal sequences are preferred which lead to GPI anchoringof the C terminus of the gene product on the cell surface, as with thehuman DAF. Alternatively, peptide sequences are preferred which controlthe membrane-compatible localisation of the gene product, as in the caseof immunoglobulins of the M isotype or of the Vesicular Stomatitis virusG protein.

According to a further preferred embodiment the virus can also containsuitable splice donor and splice acceptor sites, so that anappropriately spliced mRNA arises, which is suitable for translationwithin the individual to be treated. The nucleic acid can in additioncontain a sequence which is suitable as a ribosome binding site.

According to a further embodiment of the invention, a method for theproduction of a recombinant virus is provided, whereby the methodcomprises the steps:

a) Transfecting of a eukaryotic host cell with a transfer vector,whereby the transfer vector

i) comprises a Plasmodium falciparum MSP-1, a nucleic acid coding for afragment or a mutein thereof, whereby the mutein is differentiated byaddition, deletion, insertion, inversion or substitution of one or moreamino acids of the MSP-1 sequence, and optionally comprises the codingsequence for a selection marker; and comprises DNA sequences, which actas promoters for the transcription control of the coding sequences;

ii) the nucleic acid according to i) is flanked by MVA sequences 5′and/or 3′, whereby the sequences are suitable for the homologousrecombination with genomic MVA-DNA in the host cell;

b) infection with a virus based on MVA, preferably MVA;

c) cultivation of the host cell under conditions suitable forhomological recombination; and

d) isolation of the recombinant virus based on MVA.

Preferably, the host cell is selected from RK13 (rabbit kidney cells),BHK21 (baby hamster kidney cells) or primary CEF (chicken embryofibroblast cells).

The transfer vector can be a typical Vaccinia virus transfer vector,which for example is selected from pGS20, pSC59, pMJ601, pSC65, pSC11,pCF11 and pTKgptF1s or vectors which are derived from them; refer toMethods in Molecular Biology, Volume 62, see above, Broder and Earl,page 176 and other references mentioned in it, in particular Earl,Cooper and Moss (1991) in Current Protocols in Molecular Biology(Ausubel et al.), Wiley Interscience, New York, pages 16.15.1-16.18.10.The transfection occurs according to conditions known in the state ofthe art.

The nucleic acid can in this respect have the modifications stated forthe nucleic acid of the virus.

The nucleic acid according to i) is flanked by MVA sequences orcomplementaries of it 5′ and/or 3′; preferred flanking MVA sequences areDNA sequences in each case 5′ and 3′ of naturally occurring deletionsites in the MVA genome, e.g. deletion sites II, III or VI as describedin Meyer H., Sutter G., Mayr A. (1991), J Gen Virol 72, 1031-1038 or ascan be seen from the complete genome sequence of the MVA virus (Antoineet al. 1998, Virology 244, 365-396). Preferably the transfer vectorcomprises furthermore a selection marker such as for example anantibiotic resistance or a metabolism marker. Principally however, allselection markers known in the state of the art are comprised.

For the efficient homologous recombination the MVA-DNA sequencesflanking the nucleic acid to be inserted should exhibit a length in eachcase of at least 0.5 kb.

The host cell is transfected with the transfection vector according toknown methods. The infection with MVA occurs according to standardconditions (Staib et al., 2000).

The isolation of the recombinant MVA virus occurs based on the selectionmarker within the sequence according to alternative (i). The recombinantMVA virus can be obtained either directly from the lysate of thecultivated host cells or from the culture supernatant.

According to a further embodiment, a vaccine is made available whichcomprises:

a) the recombinant virus according to the invention; and

b) a pharmacologically compatible carrier.

Pharmacologically compatible carriers are in this respect all carriersand dilution agents known in the state of the art. If a certain type ofapplication is intended, the pharmacologically compatible carrier can beselected or modified in a known manner.

The vaccine can be administered subcutaneously, intramuscularly,intravenously, transdermally, intraperitoneally or orally. The vaccineis specified for prophylaxis and/or therapy of malaria in humans andanimals.

According to a preferred embodiment, the vaccine can furthermore containMSP-1, a fragment or a mutein thereof, which is differentiated byaddition, deletion, insertion, inversion or substitution of one or moreamino acids from the Plasmodium falciparum MSP-1 sequence, and/or anucleic acid coding it. More preferably, the MSP-1 protein is in thisrespect produced recombinantly, in particular recombinantly in E. coli.The nucleic acid coding for MSP-1 or a mutein of it is preferably onewhich is reduced with regard to its AT content. Especially preferred isa nucleic acid such as described in DE-19640817 A1, with which inparticular Plasmodium falciparum codons are replaced by human codonswithout changing the amino acid sequence.

Where the vaccine comprises both the recombinant virus as well as MSP-1,a fragment or a mutein of it or the coding nucleic acid, then thevaccine can be provided in kit form. It is therefore suitable forsimultaneous, sequential or separate administration of the twocomponents of the vaccine.

EXAMPLES

The following examples explain the invention, but do not restrict itsobject.

FIG. 1: Primary Structure of the MSP-1 Derived from the FCB-1 and MAD20.Strains of P. falciparum.

The arrows above the sequence identify the processing sites of thenative proteins (Holder et al., 1987), which divide MSP-1 into thefragments p83, p30, p38 and p42, which are anchored as complexes on theparasite surface. In the second process stage p42 is split to form p33and p19. The arrows below the illustrations designate the uniquelyoccurring endonuclease cleavage sites of the synthetic DNA sequences.

Abbreviations: SP=Signal Peptide, GA=GPI Anchor

HeLa cells were infected with rMVA-msp1d/38+42S or rMVA-msp1d/38+42A andthen fixed. Some cells were permeabilized with Triton X-100 afterfixing. The cells thus treated were incubated with mAk 5.2 as the firstantibody, which recognises a conformational epitope specific for MSP-1in the C-terminal part of the MSP-1 fragment p19 and a polyclonal serum,which recognises the ER protein Sec61beta (anti-ER marker). These werecolour labeled using Cy3 conjugated anti-mouse IgG (detects mAk 5.2) orFITC conjugated anti-rabbit IgG (detects anti-Sec61beta) and thenanalysed in the confocal microscope. If the cells were infected withrMVA-msp1d/38+42S or rMVA-msp1d/38+42A and permeabilised, then thesignal can be colocalised for MSP-1 D-38/42 (mAk 5.2) with the ERmarker. If in contrast the cells remain intact, MSP-1D-38/42 is onlydetected in the case of infection with rMVA-msp1d/38+42A on the surfaceof the infected cells. The ER marker here acts as a control for theintact condition of the cell membrane.

Abbreviations: ER=Endoplasmic Recticulum; mAK=monoclonal Antibody.

FIG. 2: Verification of MSP-1D-42 and MSP-1D-38/42 in HeLa CellsInfected with Recombinant MVA Using Immunoblot

HeLa cells were infected with rMVA-msp1d/42S, rMVA-msp1d/42A,rMVA-msp1d/38+42S or rMVA-msp1d/38+42A and incubated overnight. Samplesof the supernatant and the cellular fraction were separated usingSDS-gel electrophoresis under non-reducing conditions, transferred to aPVDF membrane and verified using mAb 5.2 primary antibodies. Onlychimera from the DAF signal sequence and the corresponding MSP-1Dfragments can be verified in the supernatants of the infected cells,whereas the intracellular expression in all cells infected withrecombinant MVA supplies a signal.

FIGS. 3A and 3B: Development of the Humoral Immune Response After ThreeImmunisations with rMVA-msp1d/42S or rMVA-msp1d/42A and One Immunisationwith MSP-1D-HX42 from E. coli.

In FIG. 3A the analysis of the humoral immune response is shown usingELISA with recombinantly produced MSP-1 D-HX42, purified from E. coli asantigen. The curves illustrate the p42 specific antibody development,measured on the OD₄₀₅=1. The mice were in each case immunised atintervals of three weeks with 10⁶ IU (first immunisation, simultaneouswith blood withdrawal S0) or 10⁸ IU (1st and 2nd boost, simultaneouswith S1 and S2) of rMVA-msp1d/42S. In addition the mice were injectedsubcutaneously after a further four weeks each with 5 μg of MSP-1D-HX42from E. coli in the incomplete Freund's adjuvant (one week after theblood withdrawal S3). S0 to S5 represent the times of the bloodwithdrawal and here the blood S0 to S3 was in each case taken atintervals of three weeks and the withdrawals of S4 and S5 occurred ineach case at intervals of four weeks. The arrows mark the times of theimmunisations. The asterisk marks the fourth immunisation withMSP-1D-HX42 from E. coli.

FIG. 3B shows the same analysis for the immunisation withrMVA-msp1d/42A.

FIG. 4: Development of the Humoral Immune Response After Immunisationswith rMVA-msp1d/S or rMVA-msp1d/83+30/38+42A in Combination withImmunisation with MSP-1D from E. coli.

The humoral immune response was determined using ELISA under applicationof recombinantly produced MSP-1D purified from E. coli as antigen. As inFIGS. 3A and 3B, the curves show the MSP-1D specific antibodydevelopment, measured on the OD₄₀₆=1.

The mice were in each case immunised at intervals of three weeks(labelled in the illustration by arrows). The immunisation strategiesallocated to the groups were composed as follows:

Gr. 1: 20 μg of MSP-1D (Day 0), 10⁸ IU of rMVA-msp1d/S (Day 21), 5 mice

Gr. 2: 20 μg of MSP-1D (Day 0), 10⁸ IU of rMVA-msp1d/A (Day 21), 5 mice

Gr. 3: 20 μg of MSP-1D (Day 0), 10⁸ IU of rMVA-msp1d/S (Day 21), 20 μgof MSP-1D (Day 42), 10 mice

Gr. 4: 20 μg of MSP-1D (Day 0), 10⁸ IU of rMVA-msp1d/S (Day 21), 10⁸ IUof rMVA-msp1d/S (Day 42), 10 mice

Gr. 5: 20 μg of MSP-1D (Day 0), 10⁸ IU of rMVA-msp1d/83+30/38+42A (Day21), 20 μg of MSP-1D (Day 42), 9 mice

Gr. 6: 20 μg of MSP-1D (Day 0), 10⁸ IU of rMVA-msp1d/83+30/38+42A (Day21), 10⁸ IU of rMVA-msp1d/83+30/38+42A (Day 42), 10 mice

Gr. 7: 20 μg of MSP-1D (Day 0), 10⁸ IU of rMVA-msp1d/A (Day 21), 20 μgof MSP-1D (Day 42), 5 mice

In the following, recombinant viruses, which lead to the production ofMSP-1 in its surface-anchored form, are labelled with “A” and thosewhich lead to the secretion of MSP-1 with “S”. TABLE 1 Complete list ofthe virus-constructs produced in the scope of the invention: rMVA-msp1drMVA-msp1f Secreted MSP-1 rMVA-msp1d/S rMVA-msp1d/83S rMVA-msp1d/83 +30S rMVA-msp1d/42S rMVA-msp1d/38 + 42S Surface- rMVA-msp1d/ArMVA-msp1f/A anchored MSP-1 rMVA-msp1d/83A rMVA-msp1f/83 + 30/38 + 42ArMVA-msp1d/83 + 30A rMVA-msp1f/38 + 42A rMVA-msp1d/42A rMVA-msp1d/38 +42A rMVA-msp1d/83 + 30/38 + 42A

The production of MSP-1 or the fragments and the localisation of theproteins in the infected cell was proven using confocal microscopy andis illustrated as an example of the infection of HeLa cells byrMVA-msp1d/38+42S and rMVA-msp1d/38+42A.

The secretion of all MSP-1 variants from cells infected with recombinantMVA was verified using immunoblot analyses of cellular supernatants andis illustrated here as an example for rMVA-msp1d/42S andrMVA-msp1d/38+42S (FIG. 2).

Then the recombinant MVA were examined in immunisation experiments onmice for their immunogenic effect with regard to the humoral immuneresponse. Here, for msp-1 recombinant MVA induced high antibody titersagainst the parasite protein which was determined using ELISA. The p42specific antibody titers and the observed, different immunisationpotential of the surface-anchored or secreted proteins produced by therecombinant MVA is illustrated as an example of immunisations withrMVA-msp1d/42S and rMVA-msp1d/42A (FIGS. 3A and 3B).

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1. A recombinant Modified Vaccinia Vaccine Ankara (MVA) virus comprising at least one nucleic acid coding for a Plasmodium falciparum merozoite surface protein-1 (MSP-1) protein or a fragment or mutein thereof.
 2. The recombinant MVA virus according to claim 1, wherein the MSP-1 protein is the MSP-1 protein of the isolate 3D7 or the MSP-1 protein of the FCB 1 strain.
 3. The recombinant MVA virus according to claim 1, wherein the fragment is selected from the fragments p83, p30, p38, p33, p 19 and p42 or combinations thereof.
 4. The recombinant MVA virus according to claim 1 wherein the mutein is differentiated from the MSP-1 sequence by addition, deletion, insertion, inversion and/or substitution of one or more amino acids.
 5. The recombinant MVA virus according to claim 1, wherein the nucleic acid coding for MSP-1 is reduced in its AT content compared to the wild type sequence.
 6. The recombinant MVA virus according to claim 1, wherein the nucleic acid coding for MSP-1 is under the control of a promoter.
 7. The recombinant MVA virus according to claim 1, wherein the nucleic acid at the 5′ end is fused with a nucleotide sequence coding for a signal peptide sequence.
 8. The recombinant MVA virus according to claim 7, wherein the signal peptide sequence controls the secretion of the gene product.
 9. The recombinant MVA virus according to claim 7, wherein the signal peptide sequence controls the localisation of the gene product relevant to the membrane.
 10. The recombinant MVA virus according to claim 7, wherein the signal sequence controls the GPI anchoring of the gene product.
 11. A method of production of a recombinant Modified Vaccinia Vaccine Ankara (MVA) virus wherein the method comprises the steps: a) transfecting a eukaryotic host cell with a transfer vector, wherein i) the transfer vector comprises a nucleic acid encoding a Plasmodium falciparum merozoite surface protein-1 (MSP-1) protein, or a fragment or a mutein thereof, wherein the mutein differs by the addition, deletion, insertion, inversion and/or substitution of one or more amino acids from the MSP-1 sequence; and optionally also comprises a selection marker; ii) the nucleic acid according to i) is flanked by MVA sequences 5′ and/or 3′, wherein the sequences are suitable for the homologous recombination in the host cell; b) infection with a virus based on MVA, preferably MVA; c) cultivation of the host cell under conditions suitable for homologous recombination; and d) isolation of the recombinant virus based on MVA.
 12. The method according to claim 11, wherein the virus is isolated from the culture supernatant or from the cultivated host cells.
 13. A vaccine comprising: a) the recombinant virus according to one of the claims 1 to 9; and b) a pharmacologically compatible carrier.
 14. The vaccine according to claim 13, further comprising: c) MSP-1, a fragment or a mutein thereof and/or a nucleic acid coding for MSP-1, or a fragment or mutein thereof.
 15. The vaccine according to claim 14, wherein the constituents a) and c) can be administered simultaneously, sequentially or separately.
 16. A method for the prophylaxis and/or therapy of malaria, the method comprising administering the recombinant virus of any one of claims 1 to
 9. 17. A method for the prophylaxis and/or therapy of malaria, the method comprising administering: i) a recombinant virus according to one of claims 1 to 8; and ii) MSP-1, a fragment or a mutein thereof and/or a nucleic acid coding for MSP-1, or a fragment or mutein thereof. 